Nuclear architecture and nuclear function appear to go hand in hand, as defects in nuclear organization are associated with aging and diseases such as cancer. We have been using budding yeast as a model system to study nuclear architecture. The yeast nucleus differs from that of higher eukaryotes in two aspects: (1) yeast lack lamins, proteins that play a major structural role in shaping the nucleus in metazoans, and (2) the yeast nuclear envelope (NE) remains intact throughout the cell cycle, unlike the NE of higher eukaryotes, which breaks down during mitosis and reassembles after chromosome segregation is completed. Nonetheless, the yeast nucleus shares important features with nuclei of higher eukaryotes: the NE has to expand during the course of the cells cycle, and the nucleus has to acquire and maintain a spherical shape and a volume proportional to cell volume. How the NE expands and what determine nuclear size and shape are questions that remain to be resolved in all systems. In the past year we've initiated a genetic screen to identify mutants that have an abnormal nuclear/cell volume ratio. This work, done in collaboration with the labs of Brenda Andrews and Mike Tyers, has led to the isolation of several dozen candidates that are currently under investigation. Our previous studies focused on a yeast strain in which the Spo7 protein was inactivated. Spo7 is a conserved regulator of phospholipids synthesis; in its absence phospholipids levels increase, leading to the expansion of the endoplasmic reticulum (ER). In yeast, this is also accompanied by expansion of certain regions of the nucleus. In particular, we were able to show that only the NE associated with the nucleolus (a sub-compartment of the nucleus) expands, whereas the rest of the nuclear membrane remains juxtaposed to the bulk of the chromatin. This led to the hypothesis that in yeast there is a nuclear tether that associates the nuclear membrane to the chromatin and resists NE expansion when phospholipid levels increase. Our subsequent studied revealed that a similar nuclear extension occurs during a mitotic arrest. We also found that phospholipid synthesis continues during a mitotic arrest and that this synthesis is necessary to form the nuclear extension. Taken together, we hypothesized that the nuclear extension is a manifestation of the cell's attempt to maintain a constant nuclear/cell volume ratio under conditions where nuclear membrane expansion exceeds cell growth. To further identify processes related to nuclear shape we searched for mutants that exhibit nuclear extensions that are not confined to the nucleolar region. This project uncovered a role for the conserved topoisomerase 2, Top 2. The mechanism by which Top 2 affects nuclear shape is currently being studied in collaboration with the lab of Jeff Bachant. Another approach to understand what regulated nuclear envelope expansion and how the nuclear envelope extension is confined to the nucleolus in mitotically arrested cells is to isolate mutants that fail to form an extension upon arresting in mitosis. We were able to isolate one such mutant in the conserved polo-like kinase, Cdc5. We showed that in cdc5 mutants the nucleus remains round during a mitotic arrest. Furthermore, we showed that this is not because the nuclear membrane does not expand, but rather because the nucleus expands isometrically. Thus, Cdc5 is needed to confine nuclear envelope expansion to the region adjacent to the nucleolus. In the course of these studies we discovered that Cdc5 also regulates cell growth. We are currently examining, in collaboration with the Larabell lab, how Cdc5 is integrated into other cell growth pathways. Finally, we found that the spo7 strain exhibits an age dependent defect in mitochondrial morphology. This is also observed in other mutants in lipid synthesis pathways. We are currently collaborating with the Amon lab to determine what leads to the formation of this abnormal mitochondrial morphology, and with the Carman lab to examine whether different mutants with abnormal mitochondrial morphology exhibit similarities in their lipid profile.